An RNA protection assay using strand specific probes identified RNA molecules that were initiated at position -92 relative to the Cap site of the human dihydrofolate reductase major transcript but transcribed from the opposite DNA strand. The upstream gene transcript was about 5 kilobase long, polyadenylated and detectable in HeLa, K562 and HL60 cells. A cDNA clone of the upstream gene was isolated. Sequence data suggested that the gene encodes a protein with a molecular weight of 11 kilodalton. A transient assay using the CAT gene as a reporter showed only 165 base pairs are sufficient for activity of either of the two opposite strand promoters. Using a new high efficient DHFR minigene, we have developed a mammalian expression system by which the co-introduced gene is amplified in a short period and is expressed at a very high level. We have already succeeded in creating CHO cell clones which overproduce the human parvovirus capsid proteins and the human immunodeficiency virus envelope protein.